The inventors of the present invention have proposed a novel isothermal nucleic acid amplification method (a method of forming a self-assembly substance of probes) which is free from enzyme (Patent Documents 1 to 4). FIGS. 1 to 3 are schematic drawings explaining a method described in Patent Document 1. For example, the method described in Patent Document 1 is a method, as illustrated in FIG. 1, which uses a pair of oligonucleotide probes 18 (Honey Comb Probe; hereinafter, referred to as HCP), each of which is constituted of three regions. The respective three regions of the first HCP 18a (an X region, a Y region, and a Z region) and the respective three regions of the second HCP 18b (an X′ region, a Y′ region, and a Z′ region) have base sequences which are complementary to each other, and, when the both probes are reacted with each other, the base sequences of the respective regions are arranged suitably so that each region in one probe hybridizes with a specific region in the other probe (FIG. 2). When a plurality of pairs of HCPs are reacted, the above idea makes it possible to form a self-assembly substance 20 (polymer) of probes through hybridization of the pair of HCPs with each other (FIG. 3). In the present description, the method of forming a polymer through the above self-assembly reaction of these oligonucleotide probes (an oligonucleotide probe alternation link self-assembly reaction) is referred to as a PALSAR method.
The method described in Patent Document 2 relates to a method of forming a more stable polymer efficiently, in which guanine or cytosine is placed at the terminal of each region in the pair of HCPs, whereby a C—G bond is formed at the terminal of each region when the pair of HCPs are hybridized with each other. In examples of Patent Document 2, there is disclosed the HCP having 20 bases in each region and having G or C at the terminal of each region.
In addition, the inventors of the present invention have found that the use of the PALSAR method contributes to enhance the sensitivity of detecting a target gene (Patent Document 5). FIG. 4 shows an example of a signal amplification method on a microplate by using the PALSAR method. As shown in FIG. 4(a), a capture probe 14 (a probe for capturing) that can capture a target nucleic acid 12 is bonded to a reaction material such as a microplate 10; then, the target nucleic acid 12 is captured by the capture probe 14 as described in FIG. 4(b); an assist probe 16 having regions each being complementary to HCPs and the target nucleic acid is added as shown in FIG. 4(c); a plurality of pairs of HCPs 18 are further added as shown in FIG. 4(e), to thereby form a self-assembly substance 20 through an alternation link self-assembly reaction; and whereby being capable of amplifying a signal.
It should be noted that the signal probe-polymer (simply referred to as a polymer, too) in the present description refers to the above-mentioned self-assembly substance formed by HCPs. In addition, the assist probe refers to the probe that can specifically bond to a target analyte to be detected and has the same base sequence partially or entirely with base sequence of one of the above pair of oligonucleotide probes. The assist probe serves for linking the target analyte and the signal probe-polymer.    Patent Document 1: JP 3267576 B    Patent Document 2: JP 3310662 B    Patent Document 3: WO 02/31192    Patent Document 4: JP 2002-355081 A    Patent Document 5: WO 03/029441